Newborns as a group are at increased risk for invasive bacterial infections and resulting sepsis. Although the majority of these infections in newborns are caused by gram-positive organisms, a variable but significant percentage of bacterial infections (about 20–40%) are due to gram-negative bacteria, particularly E. coli, Haemophilus influenzae, Klebsiella spp., and Enterobacter spp. In fact, it is the gram-negative infections that are, in some studies, associated with the highest mortality rate, which can be as high as about 40%. [Beck-Sague, C M et al., Pediatr Infect Dis J 13: 1110–116 (1994) and Stoll, B J et al., J Pediatr 129: 63–71 (1996)]
The mechanisms by which newborns are at increased risk for these bacterial infections are not currently understood. Although the neutrophil defense system is innate, there are indications that its function at birth is immature and suboptimal. Previous investigations of the activity of newborn neutrophils have demonstrated impaired adherence, chemotaxis, and phagocytosis. [Wright W C Jr. et al. Pediatrics 56: 579–584 (1975); Cairo M S, AJDC, 143:40–46 (1989); Schelonka R L et al., Sem. Perinatol., 22:2–14 (1998).] Impaired stimulus-induced adhesion and migration has been associated with decreased surface expression of L-selectin and the β2-integrin Mac-1. [Dinauer, M C, in “Hematology of Infancy & Childhood,” 5th ed., Nathan and Orkin, eds., Vol I, pp 889–967 (1998)] These findings may explain the difficulty in mobilizing neutrophils to sites of bacterial infection but do not explain the decreased phagocytic and bactericidal activity of the neutrophils of newborns.
Most studies of the microbicidal mechanism of new born neutrophils have focused on the oxidative mechanism (i.e., the phagocyte oxidase/MPO/hydroxyl radical system), with conflicting data indicating either increased or decreased capacity of this oxygen-dependent mechanism in newborns. [Dinauer, supra, and Ambruso et al., Ped Res 18:1148–53 (1984).] Despite a growing literature on antibiotic proteins and peptides, little is known about the oxygen-independent microbicidal mechanisms of newborn neutrophils. A slightly decreased content of specific (secondary) granules in the neutrophils of newborns has been documented, with an associated modest (≦2-fold) decrease in lysozyme and lactoferrin content relative to adult neutrophils. [Ambruso et al., supra.] However, the major elements of the oxygen-independent antimicrobial arsenal of neutrophil primary granules, including BPI and the defensin peptides, have not been assessed in neonates. Qing et al., Infect. Immun., 64:4638–4642 (1996), compared the lipopolysaccharide (LPS) binding of newborn neutrophils to that of adult neutrophils and reported that the newborn neutrophils have lower levels of membrane-associated 55–57 kDa and 25 kDa proteins capable of binding LPS. Although the missing proteins were not identified, the size and binding properties of the 55–57 kDa protein appeared to be similar to those of bactericidal/permeability-increasing protein (BPI) and the surface LPS receptor CD14.
The rising tide of antibiotic resistance has placed renewed emphasis on the development of agents to treat bacterial infection and its sequelae. Moreover, improved technology has led to increased survival rates for extremely ill full-term as well as premature neonates, which represent a growing population at high risk for bacterial infection. Although the replacement of neutrophils by granulocyte transfusion in newborns with sepsis has apparently been beneficial in some studies [Cairo et al., Pediatrics 74: 887–92 (1984)] this potential therapy has been complicated by difficulty in obtaining histocompatible neutrophils and by transfusion reactions.
BPI is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMNs by acid extraction combined with either ion exchange chromatography [Elsbach, J. Biol. Chem., 254:11000 (1979)] or E. coli affinity chromatography [Weiss, et al., Blood, 69:652 (1987)]. BPI obtained in such a manner is referred to herein as natural BPI and has been shown to have potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein have been reported in FIG. 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Gray et al. nucleic acid and amino acid sequences are set out in SEQ ID NOS: 1 and 2 hereto. U.S. Pat. Nos. 5,198,541 and 5,641,874 discloses recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI. Recombinant human BPI holoprotein has also been produced in which valine at position 151 is specified by GTG rather than GTC, residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG) and residue 417 is alanine (specified by GCT) rather than valine (specified by GTT).
BPI is a strongly cationic protein. The N-terminal half of BPI accounts for the high net positive charge; the C-terminal half of the molecule has a net charge of −3. [Elsbach and Weiss (1981), supra.] A proteolytic N-terminal fragment of BPI having a molecular weight of about 25 kD possesses essentially all the anti-bacterial efficacy of the naturally-derived 55 kD human BPI holoprotein. [Ooi et al., J. Bio. Chem., 262: 14891–14894 (1987)]. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays some endotoxin-neutralizing activity and only slightly detectable anti-bacterial activity against gram-negative organisms. [Ooi et al., J. Exp. Med., 174:649 (1991).] An N-terminal BPI fragment of approximately 23 kD, referred to as “rBPI23,” has been produced by recombinant means and also retains anti-bacterial activity against gram-negative organisms. [Gazzano-Santoro et al., Infect. Immun. 60:4754–4761 (1992).] An N-terminal analog of BPI, designated rBPI21 [rBPI(1–193)ala132], has been produced as described in U.S. Pat. No. 5,420,019 and Horwitz et al., Protein Expression Purification, 8:28–40 (1996). An additional N-terminal analog of BPI, designated rBPI(10–193)C132A or rBPI(10–193)ala132, has been produced as described in U.S. Pat. No. 6,013,631.
The bactericidal effect of BPI was originally reported to be highly specific to gram-negative species, e.g., in Elsbach and Weiss, Inflammation: Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven Press, Ltd. (1992). The precise mechanism by which BPI kills gram-negative bacteria is not yet completely elucidated, but it is believed that BPI must first bind to the surface of the bacteria through electrostatic and hydrophobic interactions between the cationic BPI protein and negatively charged sites on LPS. In susceptible gram-negative bacteria, BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. [Elsbach and Weiss (1992), supra]. LPS has been referred to as “endotoxin” because of the potent inflammatory response that it stimulates, i.e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to lipid A, reported to be the most toxic and most biologically active component of LPS.
BPI protein products have a wide variety of beneficial activities. BPI protein products are bactericidal for gram-negative bacteria, as described in U.S. Pat. Nos. 5,198,541, 5,641,874, 5,948,408, 5,980,897 and 5,523,288. International Publication No. WO 94/20130 proposes methods for treating subjects suffering from an infection (e.g. gastrointestinal) with a species from the gram-negative bacterial genus Helicobacter with BPI protein products. BPI protein products also enhance the effectiveness of antibiotic therapy in gram-negative bacterial infections, as described in U.S. Pat. Nos. 5,948,408, 5,980,897 and 5,523,288 and International Publication Nos. WO 89/01486 (PCT/US99/02700) and WO 95/08344 (PCT/US94/11255). BPI protein products are also bactericidal for gram-positive bacteria and mycoplasma, and enhance the effectiveness of antibiotics in gram-positive bacterial infections, as described in U.S. Pat. Nos. 5,578,572 and 5,783,561 and International Publication No. WO 95/19180 (PCT/US95/00656). BPI protein products exhibit antifungal activity, and enhance the activity of other antifungal agents, as described in U.S. Pat. No. 5,627,153 and International Publication No. WO 95/19179 (PCT/US95/00498), and further as described for BPI-derived peptides in U.S. Pat. No. 5,858,974, which is in turn a continuation-in-part of U.S. application Ser. No. 08/504,841 and corresponding International Publication Nos. WO 96/08509 (PCT/US95/09262) and WO 97/04008 (PCT/US96/03845), as well as in U.S. Pat. Nos. 5,733,872, 5,763,567, 5,652,332, 5,856,438 and corresponding International Publication Nos. WO 94/20532 (PCT/US/94/02465) and WO 95/19372 (PCT/US94/10427). BPI protein products exhibit anti-protozoan activity, as described in U.S. Pat. Nos. 5,646,114 and 6,013,629 and International Publication No. WO 96/01647 (PCT/US95/08624). BPI protein products exhibit anti-chlamydial activity, as described in co-owned U.S. Pat. No. 5,888,973 and WO 98/06415 (PCT/US97/13810). Finally, BPI protein products exhibit anti-mycobacterial activity, as described in co-owned, co-pending U.S. application Ser. No. 08/626,646, which is in turn a continuation of U.S. application Ser. No. 08/285,803, which is in turn a continuation-in-part of U.S. application Ser. No. 08/031,145 and corresponding International Publication No. WO 94/20129 (PCT/US94/02463).
The effects of BPI protein products in humans with endotoxin in circulation, including effects on TNF, IL-6 and endotoxin are described in U.S. Pat. Nos. 5,643,875, 5,753,620 and 5,952,302 and corresponding International Publication No. WO 95/19784 (PCT/US95/01151).
BPI protein products are also useful for treatment of specific disease conditions, such as meningococcemia in humans (as described in U.S. Pat. Nos. 5,888,977 and 5,990,086 and International Publication No. WO97/42966 (PCT/US97/08016), hemorrhage due to trauma in humans, (as described in U.S. Pat. Nos. 5.756,464 and 5,945.399. U.S. application Ser. No. 08/862,785 and corresponding International Publication No. WO 97/44056 (PCT/US97/08941), burn injury (as described in U.S. Pat. No. 5,494,896 and corresponding International Publication No. WO 96/30037 (PCT/US96/02349)) ischemia/reperfusion injury (as described in U.S. Pat. No. 5,578,568), and depressed RES/liver resection (as described in co-owned, co-pending U.S. application Ser. No. 08/582,230 which is in turn a continuation of U.S. application Ser. No. 08/318,357, which is in turn a continuation-in-part of U.S. application Ser. No. 08/132,510, and corresponding International Publication No. WO 95/10297 (PCT/US94/11404).
BPI protein products also neutralize the anticoagulant activity of exogenous heparin, as described in U.S. Pat. No. 5,348,942, neutralize heparin in vitro as described in U.S. Pat. No. 5,854,214, and are useful for treating chronic inflammatory diseases such as rheumatoid and reactive arthritis, for inhibiting endothelial cell proliferation, and for inhibiting angiogenesis and for treating angiogenesis-associated disorders including malignant tumors, ocular retinopathy and endometriosis, as described in U.S. Pat. Nos. 5,639,727, 5,807,818 and 5,837,678 and International Publication No. WO 94/20128 (PCT/US94/02401).
BPI protein products are also useful in antithrombotic methods, as described in U.S. Pat. Nos. 5,741,779 and 5,935,930 and corresponding International Publication No. WO 97/42967 (PCT/US7/08017).